Scope and frequency of enteric bacterial pathogens isolated
from HIV/AIDS patients and their household drinking water
in Limpopo Province
CL Obi1*, J Ramalivhana2, MNB Momba3 and J Igumbor4
College of Agriculture and Environmental Sciences, School of Agriculture and Life Sciences , University of South Africa,
287 Skinner Street, PO Box 392, Unisa 0003, Pretoria, South Africa
2
Department of Microbiology, University of Venda, P/ Bag X5050, Thohoyandou 0950, Limpopo Province, South Africa
3
Department of Water Care, Tshwane University of Technology, Acadia Campus, Pretoria, South Africa
4
University of Witswatersrand School of Public Health, Johannesburg, South Africa
1
Abstract
Although HIV/AIDS and water-borne infections, exemplified by diarrhoea, are leading causes of morbidity and mortality in
developing countries, their association has received only cursory attention. This study was therefore conducted to ascertain the
scope and frequency of potential enteric bacterial pathogens isolated from stool samples of HIV-positive and -negative individuals with and without diarrhoea as well as household drinking water of the study groups in rural communities in Limpopo
Province. A prospective study involving 330 HIV-positive individuals (200 with diarrhoea and 130 without diarrhoea) and 160
HIV-negative patients, (80 with diarrhoea and 80 without) was undertaken from August 2005 to January 2006. Stool and drinking water samples were analyzed for the presence of enteric bacterial pathogens using standard microbiological methods. Of
the 330 HIV-positive individuals, 126 (38%) and 206 (62%) were males and females respectively. HIV prevalence was mostly
common in the age group 21to 30 years. A potential enteric pathogen was isolated from all (100%) of the HIV-positive individuals with diarrhoea and 68 (52.3%) without diarrhoea (P = 0.0001). Bacteria that were significantly associated with diarrhoea
among HIV-positive patients and their household drinking water were Escherichia coli, Salmonella spp., Campylobacter spp.,
Shigella spp. and Aeromonas spp. whereas Plesiomonas shigelloides was not. The same profiles of enteric bacterial pathogens
were isolated from HIV-negative individuals but at lower frequencies (P = 0.0001). Enteric pathogens were distributed across
gender and different age strata. A notable finding was the emergence of Aeromonas spp. and Plesiomonas shigelloides in
HIV infected individuals with diarrhoea. This study provides the foremost baseline reference compendium on the scope and
frequencies of enteric bacterial pathogens isolated from stool and household drinking water samples of HIV-positive and
-negative individuals with and without diarrhoea in rural communities in the Limpopo Province.
Keywords: HIV/AIDS, household, drinking water, diarrhoea, enteric bacteria, pathogens
Introduction
Enteric bacterial pathogens are responsible for most diarrhoea
episodes worldwide. Such pathogens incriminated in diarrhoeal
cases among HIV/AIDS patients in Senegal were reportedly
19.6%, 7.6% and 4.4% for entero-aggregative Escherichia coli,
Shigella spp. and Salmonella enterica respectively (Gassama
et al., 2001). In Ethiopia the HIV/AIDS patient isolation rates
were reportedly 8.1%, 4.0% and 13.1% respectively for Salmonella, Shigella and Campylobacter (Awole et al., 2002). In South
Africa, rates of 20% for Campylobacter jejuni/coli, 16.6% for
Plesiomonas shigelloides, 13.3% for Aeromonas spp. and 10%
each for Shigella, Salmonella and Escherichia coli among HIV/
AIDS patients have been reported (Obi and Bessong, 2002).
Although HIV/AIDS patients are susceptible to the same
spectrum of enteric pathogens, which may cause diarrhoea in
immuno-competent individuals, HIV/AIDS patients require
more rigorous monitoring because of their increased susceptibility to opportunistic infections, many of which have a predilection for the gastro-intestinal tract (Fauci 1999; Lubeck et al.,
* To whom all correspondence should be addressed.
 +27 12 352-4032; cell: +27 82 422-7280; fax: +27 15 516-4552;
e-mail: [email protected]
Received 26 February 2007; accepted in revised form 26 June 2007.
Available on website http://www.wrc.org.za
ISSN 0378-4738 = Water SA Vol. 33 No. 4 July 2007
ISSN 1816-7950 = Water SA (on-line)
1993). The linkage between HIV/AIDS and water may appear
super­ficial but is indeed an in-depth association and water-borne
bacterial pathogens are therefore strongly associated with HIV/
AIDS (Obi et al., 2006). Despite this correlation, there are no
available data on the scope and frequency of enteric bacterial
pathogens isolated from HIV/AIDS patients and their household
drinking water.
The aim of this study was to determine the scope and frequency of enteric bacterial pathogens isolated from HIV/AIDS
patients with and without diarrhoea and their household drinking water in Limpopo Province.
Materials and methods
Study area
Areas for the collection of samples were stratified according to
the 6 districts in Limpopo Province (Oni et al., 2002). Three
districts were selected for the study based on familiarity with the
area, HIV prevalence and other parameters. The selected districts comprised Vhembe, Waterberg, and Capricorn.
The sites in Vhembe such as Musina represent the northernmost
part of Limpopo Province and are located in the Limpopo valley. It
is the main gateway to South Africa from the north and Musina is
also a border town linking South Africa and Zimbabwe.
539
In the Waterberg district Bela-Bela area was chosen to
represent the Bushveld region of the province. Bela-Bela is an
important tourist centre and holiday resort town, with proximity
to the Gauteng region. Waterberg district is also known to have a
relatively higher HIV prevalence (26.6%) when compared to the
provincial average (19.3%) (DOH, 2005). Mankweng area was
selected to represent the Capricorn region or the central region
of the Limpopo Province.
Prior to the commencement of the study, preliminary visits were undertaken to each of the chosen study areas by members of the research team. During these visits, the background,
protocols, objectives and potential significance of the study
including issues around confidentiality and consent were discussed with care-givers, support groups and non-governmental
organisations and their support were sought before collection
of specimens. The study team chose to work closely with support groups, NGOs and HIV care-givers because they provide
a ‘comfort zone’ for HIV/AIDS patients, who in turn confide in
the care-givers. Due to the stigmatisation of the disease, identifying HIV/AIDS patients is usually an uphill task in most South
African communities. Health authorities and family members
are usually hesitant to divulge information on HIV/AIDS status of individuals because of ethical issues and the pressure to
maintain confidentiality. The support groups, care-givers and
NGOs who work directly with HIV/ADIS patients provided the
platform to reach out to the participants.
bacterial analyses within 4 to 6 h of collection. Water samples
were collected in sterile containers from the same households
where stool specimens were collected and transported in cooler
boxes to the same laboratory for bacteriological analyses within
4 to 6 h of collection.
Culture media
Isolation media commonly used for the isolation of enteric bacterial pathogens were employed in this study. They consisted of
MacConkay agar (MCA), Shigella-Salmonella agar (S-S agar),
Xylose deoxycholate citrate agar (XDCA), Thiocitrate bile salt
(TCBS) agar, Kleigler’s iron agar (KIA), enrichment broths and
alkaline peptone water.
Isolation of bacterial enteric pathogens
The present study was conducted between August 2005 to January 2006. Information on age, gender, diarrhoeal and HIV status
was obtained.
HIV-positive and HIV-negative individuals were enlisted in
this study. The study group consisted of 330 HIV-positive individuals made up of 200 HIV-positive individuals with diarrhoea and
130 HIV-positive individuals without diarrhoea. Similarly, a total
of 160 HIV-negative individuals consisting of 80 HIV-negative
individuals with diarrhoea and another 80 HIV-negative individuals without diarrhoea were also included as unmatched controls
A diarrheic stool in this study was regarded as the passage
of loose or watery stools. Stool samples from HIV-positive and
-negative individuals with and without diarrhoea were analysed
for the presence of potential bacterial pathogens.
All cases and controls had stool and water specimens collected
and processed in the same manner. For the isolation of Aeromonas and Plesiomonas species, incubation of seeded XDCA
agar plates was at 37OC for 24 h. Colonies were screened for
oxidase production and oxidase positive colonies were identified
as belonging to the genera Aeromonas and Plesiomonas using a
battery of biochemical tests (Sinha et al., 2004; Obi et al., 1995;
1998) and also confirmed using API 20E (Analytab product). For
the isolation of Campylobacter, specimens were plated on Butzler’s media and the inoculated plates were incubated under a
micro-aerophilic atmosphere (Campy Pak, BBL, Microbiology
Systems, Cockeysville, Md) at 42OC for 72 h. One typical colony
was selected and identified by testing for Gram-stain reaction,
microscopic cell morphology, catalase and oxidase production.
Campylobacter jejuni and coli were separated based on hydrolysis of hippurate and indoxyl acetate. C. jejuni is positive for both
tests whereas C. coli only hydrolyses indoxyl acetate (Prasad et
al., 1994)
Schemes for the isolation of Salmonella and Shigella species
included primary isolation on DCA or S-S agar and subculturing of suspected colonies on KIA and testing for motility urea
hydrolyses and indole production. Selenite F broth was used to
enhance recovery of Salmonella and Shigella (Farmer, 1995).
For the detection of Vibrio species, specimens were plated on
thiocitrate bile salt sucrose medium and enriched with alkaline
peptone water.
Ethics approval
Statistical analysis
The Health, Safety and Ethics Committee of the University of
Venda, Thohoyadou, South Africa granted ethical approval for
this study. Informed consent was obtained from study subjects
before collection of stool and water samples. Issues of confidentiality and anonymity were also maintained.
Graphs and tables were used for data presentation. Statistical
differences between the proportions of enteric pathogen isolates
in the case and control groups were established with chi-square
test. Spearman’s rho correlation was used to assess the strength
of association between enteric pathogen isolates from respective
stool and water samples of the various case and control groups.
Study population
HIV testing
Screening for HIV sero-status was preformed using the
OraQuick HIV1 and HIV2 (Ora Sure Technology, USA) test kit
as described by the manufacturers and as previously reported
(Obi and Bessong, 2002).
Collection and transportation of stool and water
samples
Stool specimens were collected in clean, sterile wide-mouth
containers and transported in cooler boxes to the laboratory for
540
Results
Demographic characteristics of study participants
A total of 330 HIV-positive individuals comprising 200 HIVpositive individuals with diarrhoea and 130 HIV-positive individuals without diarrhoea were enrolled in the study. Similarly,
160 HIV-negative individuals comprising 80 each of HIV-negative individuals with and without diarrhoea respectively were
also enrolled. The age and gender distribution of the study participants are presented in Table 1.
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TABLE 1
Gender and age strata of HIV-positive and -negative individuals with and without diarrhoea
Gender
Male
Female
0-10
11-20
Age group
21-30
31-40
41-50
>50
HIV-positive Individuals (n = 330)
Total
HIV-negative individuals (n = 160)
HIV negative with diarrhoea (n = 80)
HIV Negative without diarrhoea (n = 80)
Total
From the 330 HIV-positive individuals, 126 (38%) and 206 (62%) were males
and females respectively. HIV infection
was predominant in the age group 21 to 30
(31.5%) and in the age brackets 11 to 20
years and 31 to 40 years (Table 1).
Prevalence of enteric pathogens
across study groups
Figure 1 shows the prevalence rates of
potential bacterial diarrhoea agents from
HIV-positive and -negative individuals
with and without diarrhoea. A potential
enteric bacterial pathogen was isolated
from all (100%) of HIV-positive individuals with diarrhoea and 68 controls without
diarrhoea (52.3%) (P = 0.0001)
Bacteria that were significantly associated with diarrhoea were Escherichia
coli, Salmonella spp., Campylobacter spp.,
Shigella and Aeromonas spp. whereas
P. shigelloides was not. Yersinia spp. and
Vibrio spp. were not isolated from HIVpositive individuals with diarrhoea.
The same type of enteric bacterial
pathogens was also isolated from HIVnegative individuals with and without
diarrhoea but at lower frequencies (P =
0.0001).
Shigella spp. (26.5%) and Aeromonas
(20%) were more prevalent among HIVnegative individuals (Fig. 2). Profiles of
enteric pathogens from household drinking water of HIV-positive study participants with and without diarrhoea also
showed that Escherichia coli (EPEC),
Salmonella spp., Campylobacter spp.,
Shigella and Aeromonas spp. were isolated at comparable frequencies as in the
respective study groups (Fig. 3).
114
(57.0%)
90
(69.0%)
204
(62.0%)
8
(4.0%)
4
(3.0%)
12
(3.6%)
38
(19.0%)
26
(20.0%)
64
(19.4%)
60
(30.0%)
44
(33.9%)
104
(31.5%)
39
(19.5%)
24
(18.5%)
63
(19.1%)
29
(14.5%)
23
(17.7%)
52
(15.8%)
26
(13.0%)
9
(6.9%)
35
(10.6%)
36
(45.0%)
33
(41.3%)
69
(43.0%)
44
(55.0%)
47
(58.7%)
91
(57.0%)
5
(6.3%)
4
(5%)
9
(5.6%)
19
(23.8%)
21
(26.3%)
40
(25.0%)
20
(25.0%)
16
(20%)
36
(22.5%)
14
(17.5%)
19
(23.8%)
33
(20.6%)
12
(15.0%)
9
(11.3%)
21
(13.1%)
10
(12.5%)
11
(13.8%)
21
(13.1%)
E.coli (EPEC)
Salmonella spp
Campylobacter
spp
Shigella spp
Aeromonas
P.shigelloides
Other
(Enterococcus)
25.0%
20.0%
Prevalence (%)
HIV positive without diarrhoea (n = 130)
86
(43.0%)
40
(31.0%)
126
(38.0%)
15.0%
10.0%
5.0%
0.0%
Pathogen
HIV + individuals with diarrhoea
HIV + individuals without diarroea
Figure 1
Isolation of potential bacterial diarrhoea agent from HIV-positive individuals
with and without diarrhoea
30.0%
25.0%
20.0%
Prevalence (%)
HIV positive with diarrhoea (n = 200)
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15.0%
10.0%
5.0%
0.0%
E.coli (EPEC)
Salmonella spp
Campylobacter
spp
Shigella spp
HIV - individuals with diarrhoea
Aeromonas
P.shigelloides
Pathogen
Other
(Enterococcus)
HIV - individuals without diarroea
Figure 2
Isolation of potential bacterial diarrhoea agents from HIV-negative individuals
with and without diarrhoea
541
Data on enteric bacterial pathogens from HIV-positive and -negative individuals with diarrhoea
accordingly to gender showed a
significant association with diarrhoea for all pathogens except
Yersinia and Vibrio spp. Age distribution showed that enteropathogens were isolated from virtually
all age groups (Table 2). For the
control group (without diarrhoea)
isolation according to gender and
age groups was similar but with
lower frequencies (Table 3).
30.0%
25.0%
20.0%
15.0%
10.0%
5.0%
0.0%
E.coli
Salmonella spp
Campylobacter
spp
Shigella spp
Aeromonas
P.shigelloides
Isolation rate from water sources of HIV + individuals with diarrhoea
Isolation rate from water sources of HIV + individuals without diarrhoea
Figure 3
Profiles of enteric bacterial pathogens isolated from household drinking water of
HIV-positive individuals with and without diarrhoea
TABLE 2
Gender and age distribution of enteric bacterial pathogens isolated from HIV-positive
and HIV-negative individuals with diarrhoea
Bacterial pathogens
A) HIV-positive individual
Escherichia coli (n = 43)
Salmonella (n = 38)
Campylobacter (n = 41)
Shigella (n = 27)
Aeromonas hydrophila (n = 30)
Plesiomonas (n =16)
Enterococcus (n = 5)
Yersinia spp. (n = 0)
Vibrio spp. (n = 0)
B) HIV-negative individuals
Escherichia coli (n =10)
Salmonella (n =10)
Campylobacter (n =14)
Shigella (n =21)
Aeromonas hydrophila (n =16)
Plesiomonas (n =5)
Enterococcus: (n =4)
Yersinia spp. (n = 0)
Vibrio spp. (n = 0)
Gender
Male
Female
0-10
Age group
21-30
31-40
15 (34.9%)
16 (42.1%)
20 (48.8%)
15 (55.6%)
13 (43.3%)
6 (37.5%)
3 (60.0%)
0 (0.0%)
0 (0.0%)
28(65.1%)
22 (57.9%)
21 (51.2%)
12 (44.4%)
17 (56.7%)
10 (62.5%)
2 (40.0%)
0 (0.0%)
0 (0.0%)
2 (4.7%)
1 (2.6%)
3 (7.3%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
6 (60.0%)
3 (30.0%)
5 (35.7%)
9 (42.9%)
7 (43.8%)
2 (40.0%)
3 (75.0%)
0 (0.0%)
0 (0.0%)
4 (40.0%)
7 (70.0%)
9 (64.3%)
12 (57.1%)
9 (56.3%)
3 (60.0%)
1 (25.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%) 3 (30.0%) 4 (40.0%)
4 (40.0%) 6 (60.0%) 0 (0.0%)
0 (0.0%)
8 (57.1%) 4 (28.6%)
0 (0.0%) 6 (28.6%) 2 (9.5%)
0 (0.0%) 7 (43.8%) 0 (0.0%)
0 (0.0%) 5 (100.0%) 0 (0.0%)
2 (50.0%) 0 (0.0%) 2 (50.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
Comparison of proportions of enteric pathogens
isolated from stool and water samples of study
participants
Figures 5 and 6 show the prevalence of enteric pathogens isolated
from stool and water samples respectively for HIV-positive and
HIV-negative individuals with diarrhoea. Shigella spp. (26.25%)
were the most prevalent enteric pathogens isolated from the stool
samples of HIV-negative individuals with diarrhoea followed by
Aeromonas spp. (20.00%) and Campylobacter (17.50%). Among
HIV-positive individuals with diarrhoea, E. coli (21.50%) was
most prevalent followed by Campylobacter (20.50%). P. shigel-
542
11-20
8 (18.6%) 14 (32.6%) 9 (20.9%)
3 (7.9%) 12 (31.6%) 11 (29%)
16 (39.0%) 11 (26.8%) 7 (17.1%)
9 (33.3%) 9 (33.3%) 5 (18.5%)
11 (36.7%) 9 (30.0%) 7 (23.3%)
4 (25.0%) 4 (25.0%) 3 (18.8%)
4 (80.0%) 1 (20.0%) 0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
1 (7.1%)
6 (28.6%)
3 (18.8%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
41-50
>50
7 (16.3%)
6 (15.8%)
4 (9.8%)
3 (11.1%)
3 (10.0%)
3 (18.8%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
3 (7.0%)
5 (13.2%)
0 (0.0%)
1 (3.7%)
0 (0.0%)
2 (12.5%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
3 (30.0%)
0 (0.0%)
1 (7.1%)
3 (14.3%)
4 (25.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
4 (19.1%)
2 (12.5%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
loides was the least commonly isolated enteric pathogen in both
HIV-positive (8.0%) and HIV-negative individuals (6.25%). The
relative risk (which is the ratio of HIV infected individuals with
diarrhoea to HIV-negative individuals with diarrhoea) was highest with E. coli (1.7) followed by Salmonella (1.5), P. shigelloides
(1.3) and Campylobacter (1.2).
Higher percentages of enteric pathogens were isolated from
household drinking water of HIV-negative individuals with
diarrhoea when compared to percentages isolated from household drinking water of HIV-positive individuals. Among HIVnegative individuals Salmonella (37.50%) was the most prevalent isolate followed by Shigella (25.0%). Similarly, Salmonella
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TABLE 3
Gender and age distribution of enteric bacterial pathogens isolated from HIV-positive
and HIV-negative individuals without diarrhoea
Bacterial pathogens
A) HIV-positive individual
Escherichia coli (n = 13)
Salmonella (n = 16)
Campylobacter (n = 11)
Shigella (n = 5)
Aeromonas hydrophila (n = 7)
Plesiomonas (n =6)
Enterococcus: (n = 10)
Yersinia spp. (n = 0)
Vibrio spp. (n = 0)
B) HIV-negative individuals
Escherichia coli (n =3)
Salmonella (n =5)
Campylobacter (n =9)
Shigella : HIV-WD (n =6)
Aeromonas hydrophila (n =6)
Plesiomonas (n =4)
Enterococcus (n = 3)
Yersinia spp. (n = 0)
Vibrio spp. (n = 0)
Gender
Male
Female
0-10
11-20
Age group
21-30
31-40
41-50
>50
5 (38.5%)
7 (43.8%)
6 (54.6%)
2 (40%)
4 (57.1%)
2 (33.3%)
4 (40.0%)
0 (0.0%)
0 (0.0%)
8 (61.5%)
9 (56.3%)
5 (45.5%)
3 (60.0%)
3 (42.9%)
4 (66.7%)
6 (60.0%)
0 (0.0%)
0 (0.0%)
0 (0.00%)
2 (12.5%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
1 (10.0%)
0 (0.0%)
0 (0.0%)
3 (23.1%)
5 (31.3%)
4 (36.4%)
1 (20.0%)
2 (28.6%)
2 (33.3%)
6 (60.0%)
0 (0.0%)
0 (0.0%)
7 (53.9%)
6 (37.5%)
5 (45.5%)
3 (60.0%)
3 (42.9%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
3 (23.1%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
3 (30.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
3 (18.8%)
0 (0.0%)
1 (20.0%)
0 (0.0%)
4 (66.7%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
2 (18.2%)
0 (0.0%)
2 (28.6%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
2 (66.7%)
2 (40.0%)
4 (44.4%)
2 (33.3%)
3 (50.0%)
2 (50.0%)
3 (100.0%)
0 (0.0%)
0 (0.0%)
1 (33.3%)
3 (60.0%)
5 (55.6%)
4 (66.7%)
3 (50.0%)
2 (50.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
1 (16.7%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
3 (60.0%)
0 (0.0%)
2 (33.3%)
4 (66.7%)
3 (75.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
2 (40.0%)
0 (0.0%)
3 (50.0%)
0 (0.0%)
2 (50.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
2 (33.3%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
40.0%
35.0%
Prevalence (%)
30.0%
Figure 4
Profiles of enteric bacterial pathogens isolated from household
drinking water of HIV-negative
individuals with and without diarrhoea
25.0%
20.0%
15.0%
10.0%
5.0%
0.0%
E.coli
Salmonella spp
Campylobacter
spp
Shigella spp
Aeromonas
P.shigelloides
Pathogen
HIV - individuals with diarrhoea
HIV - individuals without diarrhoea
Figure 5: Profiles of enteric bacterial pathogens isolated from HIV-positive and -negative individuals with
diarrhoea
30.0%
25.0%
20.0%
Prevalence (%)
Figure 5
Profiles of enteric bacterial
pathogens isolated from HIVpositive and -negative individuals with diarrhoea
15.0%
10.0%
5.0%
0.0%
E.coli
Salmonella spp
Campylobacter
spp
Shigella spp
Aeromonas
P.shigelloides
Others
Pathogen
HIV + individuals with diarrhoea
HIV - individuals without diarrhoea
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543
40.0%
35.0%
Figure 6
Profiles of enteric bacterial
pathogens isolated from water
samples of HIV-positive and
-negative individuals with
diarrhoea
Prevalence (%)
30.0%
25.0%
20.0%
15.0%
10.0%
5.0%
0.0%
E.coli
Salmonella spp
Campylobacter spp
Shigella spp
Pathogen
HIV + individuals with diarrhoea
(24.50%) was the most prevalent enteric pathogens isolated from
household drinking water of HIV-positive individuals. P. shigelloides was the least isolated organism from household water
sources of both groups.
Further to the above figures, the chi-square analyses below
try to establish whether the differences in proportion of enteric
pathogens isolated from stool and water samples of the case and
control groups differed significantly.
E. coli, Salmonella, Campylobacter and P. shigelloides
isolated from the stool samples exhibited very similar patterns (Tables 4 to 7). In both comparisons (HIV-negative individuals without diarrhoea vs. HIV-positive individuals without
diarrhoea; and HIV-negative with diarrhoea vs. HIV-positive
individuals with diarrhoea), higher proportions of the enteric
pathogens were isolated from stool samples of HIV-positive individuals with and without diarrhoea. However, only the comparison of prevalence of E. coli across HIV-positive and -negative
individuals without diarrhoea reached statistical significance
Aeromonas
P.shigelloides
HIV - individuals without diarrhoea
(p = .018) (Table 4). In this instance, E. coli was significantly
more common among HIV-positive individuals without
diarrhoea.
Shigella and Aeromonas (Tables 8 and 9) were, however,
more prevalent in the stool samples of HIV-negative individuals
(with or without diarrhoea) when compared to the HIV-positive
individuals (with or without diarrhoea), but these were not statistically significant except for the comparison of the prevalence
of Shigella species across HIV-negative with diarrhoea vs. HIVpositive individuals with diarrhoea (p = 0.011) (Table 9). In this
comparison, Shigella species were significantly more prevalent
among HIV-negative individuals with diarrhoea.
For the water samples, HIV-negative individuals had higher
percentages of all 6 enteric pathogens (E. coli, Salmonella,
Campylobacter, Shigella, Aeromonas and P. shigelloides) in
their household drinking water when compared with percentages isolated from water samples from households of HIV-positive individuals (Tables 4 to 9). In these comparisons ((1) HIV
TABLE 4
Chi-square analysis of E. coli isolated from stool and household drinking water by HIV status
and presence or absence of diarrhoea
Health status
HIV-negative individuals without diarrhoea
HIV-positive individuals without diarrhoea
HIV-negative individuals with diarrhoea
HIV-positive individuals with diarrhoea
* Significant at the 0.05 level (2-sided)
E. coli in stool
Count (percentage)
Significance
3 (3.8%)
18 (13.8%)
10 (12.5)
43 (21.5%)
.018*
.083
E. coli in household drinking water
Count (percentage)
Significance
7 (8.8%)
9 (6.9%)
18 (22.5%)
29 (14.5%)
.628
.106
Table 5
Chi-square analysis of Salmonella species isolated from stool and household drinking water
by HIV status and presence or absence of diarrhoea
Salmonella spp. in stool
Health status
HIV-negative individuals without diarrhoea
HIV-positive individuals without diarrhoea
HIV-negative individuals with diarrhoea
HIV- positive individuals with diarrhoea
Count (percentage)
Significance
5 (6.3%)
16 (12.3%)
11 (13.8%)
38 (19.0%)
.155
.296
Salmonella spp. in household drinking
water
Count (percentage)
Significance
10 (12.5%)
18 (13.3%)
30 (37.5%)
49 (24.5%)
.780
.029*
* Significant at the 0.05 level (2-sided)
544
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Table 6
Chi-square analysis of Campylobacter species isolated from stool and household drinking water by
HIV status and presence or absence of diarrhoea
Campylobacter spp. in stool
Health status
HIV- negative individuals without diarrhoea
HIV- positive individuals without diarrhoea
HIV- negative individuals with diarrhoea
HIV- positive individuals with diarrhoea
Count (percentage)
9 (11.3%)
11 (8.5%)
14 (17.5%)
41 (20.5%)
Significance
.504
.568
Campylobacter spp. in household
drinking water
Count (percentage)
Significance
8 (10%)
14 (10.8%)
19 (23.8%)
33 (16.5%)
.860
.159
* Significant at the 0.05 level (2-sided)
TABLE 7
Chi-square analysis of Plesiomonas species isolated from stool and household drinking water by
HIV status and presence or absence of diarrhoea
Plesiomonas spp. in stool
Health status
HIV-negative individuals without diarrhoea
HIV-positive individuals without diarrhoea
HIV-negative individuals with diarrhoea
HIV-positive individuals with diarrhoea
Count (percentage)
4 (5.0%)
7 (5.4%)
5 (6.3%)
16 (8.0%)
Significance
.903
.615
Plesiomonas spp. in household
drinking water
Count (percentage)
Significance
2 (2.5%)
2 (1.5%)
6 (7.5%)
10 (5.0%)
.621
.416
* Significant at the 0.05 level (2-sided)
Table 8
Chi-square analysis of Shigella species isolated from stool and household drinking water by
HIV status and presence or absence of diarrhoea
Shigella spp. in stool
Health status
HIV-negative individuals without diarrhoea
HIV-positive individuals without diarrhoea
HIV-negative individuals with diarrhoea
HIV-positive individuals with diarrhoea
* Significant at the 0.05 level (2-sided)
Count (percentage)
6 (7.5%)
5 (3.8%)
21 (26.3%)
27 (13.5%)
Significance
.248
.011*
Shigella spp. in household drinking
water
Count (percentage)
Significance
8 (10.0%)
12 (9.2%)
20 (25.0%)
20 (10.0%)
.845
.001*
Table 9
Chi-square analysis of Aeromonas species isolated from stool and household drinking water by
]HIV status and presence or absence of diarrhoea
Aeromonas spp. in stool
Health status
HIV-negative individuals without diarrhoea
HIV-positive individuals without diarrhoea
HIV-negative individuals with diarrhoea
HIV-positive individuals with diarrhoea
* Significant at the 0.05 level (2-sided)
Count (percentage)
Significance
6 (7.5%)
7 (5.4%)
16 (20.0%)
30 (15.0%)
537
negative without diarrhoea vs. HIV-positive individuals without
diarrhoea and (2) HIV negative with diarrhoea vs. HIV-positive
individuals with diarrhoea), HIV-negative individuals with diarrhoea had significantly higher proportions of Salmonella (p =
.029) (Table 5), Shigella (p = .001) (Table 8) and Aeromonas (p =
.014) (Table 9) in their water sources when compared with proportions isolated from their respective HIV-positive individuals
with diarrhoea cohorts.
The Spearman’s correlation analysis below established a
strong correlation between the proportions of enteric pathogens
isolated from water samples with proportions isolated from stool
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.308
Aeromonas spp. in household drinking
water
Count (percentage)
Significance
6 (7.5%)
13 (10.0%)
19 (23.8%)
24 (12.0%)
.540
.014*
samples. For all the organisms and cohort groups the level of significance was at 0.000. The strength of association exceeded 0.7
in most of the cases but varied with organism and cohort group
with no consistent pattern (Table 10)
Discussion
This study provides the first baseline reference data on the scope
and frequency of enteric bacterial pathogens isolated from the
stool samples and household drinking water of HIV-positive and
-negative individuals with and without diarrhoea in rural com-
545
Table 10
Spearman’s rho correlation of respective enteric pathogen isolated in stool and household drinking water
by HIV status and presence or absence of diarrhoea
Organism
E. coli
Salmonella
Campylobacter
Shigella
Aeromonas
Plesiomonas spp.
Total number
Correlation coefficient
Significance
Correlation coefficient
Significance
Correlation coefficient
Significance
Correlation coefficient
Significance
Correlation coefficient
Significance
Correlation coefficient
Significance
* Significant at the 0.05 level (2-sided)
HIV-negative
individuals without diarrhoea
.637(**)
.000
.683(**)
.000
.936(**)
.000
.854(**)
.000
1.000(**)
.
.698(**)
.000
80
munities in Limpopo Province. Inclusion of controls has made it
possible to ascertain any epidemiological linkage of pathogens
studied with diarrhoea, water sources and HIV/AIDS.
Gender distribution among HIV-positive patients, consisting of 62% infection rate in women as opposed to 38% in males
(P = 0.0001) confirms previous reports on feminisation of the
epidemic (UNAIDS, 2006) Feminisation has been attributed to
unequal power relations between men and women, and increasing vulnerability of women due to unemployment and poverty, which drives them to prostitution as a means of survival.
Age distribution of HIV-positive individuals with and without
diarrhoea indicated that infection burden was common to all
age groups studied but mostly in the 21 to 30 years age group
(31.5%), followed by the age groups 11 to 20 (19.4%) and 31 to
40 (19.1%). The pooled affected age group (11 to 40 years) constitutes the most productive range of any economy. The ripple
effects of these observations on the various sectors of the economy including water resource management will be increasingly
devastating.
The high frequency of isolating Escherichia coli, Salmonella
species, Campylobacter jejuni/coli and Aeromonas species from
HIV-positive individuals with diarrhoea in contrast with the low
frequency of isolation from those without diarrhoea strongly
incriminates the pathogens in diarrhoeal cases of HIV-positive
individuals. These findings agree with the reports of Weiss et
al. (2005) from Lima, Peru. They identified one or more enteric
pathogens in 53% of case subjects and 21% of control subjects.
In a related study on enteric pathogens in Southern Indian HIV
infected patients with and without diarrhoea, enteric pathogens
were detected from stool samples in 57.4% of diarrhoeal patients
compared to 40% of those without diarrhoea (P>0.05) (Mukhopadhya et al., 1999). Specifically, bacterial pathogens were isolated more commonly from patients with diarrhoea (12/61) compared to patients without diarrhoea (2 /50) (P < 0.05).
Another interesting aspect of the present study was the
observation on the differences in main bacterial aetiologies
of diarrhoea according to HIV status of patients. In immuno suppressed HIV-positive individuals with diarrhoea, the leading bacterial aetiologies were enteropathogenic E. coli (21.5%),
Campylobacter jejuni/ coli (20.5%), Salmonella species (19%)
546
HIV-positive
individuals without diarrhoea
.680(**)
.000
.935(**)
.000
.875(**)
.000
.627(**)
.000
.716(**)
.000
.524(**)
.000
130
HIV-negative
individuals with
diarrhoea
.701(**)
.000
.515(**)
.000
.825(**)
.000
.968(**)
.000
.896(**)
.000
.907(**)
.000
80
HIV-positive
individuals with
diarrhoea
.787(**)
.000
.850(**)
.000
.875(**)
.000
.844(**)
.000
.879(**)
.000
.778(**)
.000
200
and Aeromonas species (15%). In immuno-competent individuals, Campylobacter (17.5%), and Shigella species (26%) were
the main pathogens although Gassama et al., (2001) reported
Shigella and Salmonella for immuno-competent individuals,
and E.coli, Shigella and Salmonella for immuno-compromised
individuals. HIV-infected patients are usually susceptible to the
same spectrum of clinical manifestations and enteric bacterial
pathogens implicated in diarrhoeal cases in HIV-negative individuals (Weber et al., 1999; Wilcox, 2000) and this was variously observed in this study. Salmonella gastro-enteritis usually
manifests as watery diarrhoea, abdominal pain, fever, nausea,
vomiting or as an enteric fever. Shigella and Campylobacter
usually show as dysentery marked by muco-purulent bloody
diarrhoea and fever (Wilcox, 2000). In addition to stool culture,
routine blood tests may reveal the severity of the diarrhoea, such
as the state of dehydration and electrolyte profiles. A highly elevated leukocyte count may be suggestive of bacterial colitis or
a complication such as perforation or intra-abdominal abscess
formation.
The present investigation has revealed the frequent association of Aeromonas spp. in cases of diarrhoea in HIV patients.
However, the role of Aeromonas spp. as a diarrhoeagenic bacterial pathogen is still regarded by some investigators as controversial (Albert et al., 1999). Aeromonas spp. have been associated with diarrhoea in some studies (Pazzaglia et al., 1991; Obi
et al., 1995). Albert et al. (1999) asserted that no well-described
epidemiologically linked outbreaks of Aeromonas-induced diarrhoea have been reported. Plesiomonas shigelloides was not
strongly associated with diarrhoea in HIV-positive individuals
studied despite its incrimination in several case reports of diarrhoea and numerous diarrhoea cases linked to the organism (Obi
et al., 1998; Brenden et al., 1988).
Diarrhoea has assumed an elevated status among HIVpositive patients because of the chronicity of diarrhoea in
HIV-infected patients. Chronic diarrhoea results in substantial
morbidity and mortality, reduced quality of life and increased
health-care burden (Lubeck et al., 1993). Although CD4 counts
were not included in this study, previous studies showed that
CD4 counts were lower in patients with diarrhoea, corroborating
the assertions that diarrhoea is a debilitating medical condition
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and that effective management can prevent immuno suppression
(Gassama et al., 2001).
Another observation in this study was the infection with multiple pathogens, indicating that pathogens may act in synergy to
induce diarrhoea. Reports on the isolation of mixed pathogens
from diarrheic stool samples abound in the literature (Faruque
et al., 1994; Albert et al., 1999). Interestingly, Yersinia spp. and
Vibrio cholerae were not isolated from HIV-positive and -negative individuals with and without diarrhoea in the present study.
It may therefore be concluded that both pathogens may be rare
aetiological agents of diarrhoea in rural communities in Limpopo
Province. Schemes for the isolation of Clostridium difficile, a
notable cause of antibiotic associated diarrhoea and pseudo-membranous colitis (Albert et al., 1999) were not included in this study.
No attempts were made to isolate viral and parasitic agents.
The scope and frequencies of enteric bacterial pathogens
isolated from household drinking water of HIV-positive individuals were not significantly different from those isolated from
their stool samples. The cankerworm of HIV and water-borne
infections is bound to be rapidly debilitating and fatal.
This is compounded by hygiene and sanitation issues. The
majority of the households studied had no proper water storage containers. Water storage containers had no lids, were dirty
and not regularly washed. General personal and environmental
hygiene was poor, toilet facilities were buzzing with flies and
hand-washing after using toilet facilities was not common. In
some cases, faeces were not properly disposed of as they were
discharged into gutters or into surrounding veld. Drainage systems were poor in the majority of the study areas.
Mitigating factors should include unrelenting efforts to destigmatise HIV infections, continuous public awareness and
educational campaigns on the relationship between HIV/AIDS
and water quality and the various methods for household treatment and storage of water, including sanitation and hygiene.
Educational campaigns should also target caregivers, lay counsellors and household family members.
Management of diarrhoeal cases may be predicated on the
use of antibiotics because antibiotics have been reported to
shorten the duration of diarrhoea, decrease stool output and
abrogate some complications. Determination of antibiotic susceptibility profiles of isolated pathogens is therefore crucial and
is indeed the focus of an extended investigation. Another interesting focus of an extended study is to undertake genetic studies
to unravel any relatedness between enteric bacterial pathogens
from water sources and stool samples.
In conclusion, the present study has revealed E. coli, Salmonella, Campylobacter and Aeromonas as the major pathogens incriminated in diarrhoeal cases among HIV-positive
individuals and their household drinking water. Other findings
include low prevalence of P. shigelloides and lack of isolation of
Yersinia spp. and Vibrio cholerae from diarrheic cases and the
seeming synergistic action of the pathogens in the induction of
diarrhoea. A notable finding was the emergence of Aeromonas
spp. in diarrhoeal cases of HIV-positive individuals and their
household drinking water. This study is the first report on the
scope and frequency of enteric bacterial pathogens isolated from
HIV/AIDS patients and their household drinking water in Limpopo Province, South Africa.
Acknowledgement
The authors are very grateful to the Water Research Commission
(WRC) for providing funds for the project and to the National
Research Foundation (NRF) for student support through post-
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graduate student bursaries. We appreciate the various contributions of HIV/AIDS support groups and non-Governmental
organisations (NGOs) in the execution of this project.
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